Dihydropyridine-3,5-dicarboxylic acid ester derivatives

ABSTRACT

A dextro-rotatory optical isomer of diastereoisomer A of (±)-2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid 3-(1-benzylpyrrolidin-3-yl)ester 5-methyl ester is provided having the formula ##STR1## and wherein the melting point of the hydrochloride of the dextro-rotatory optical isomer is 223° to 230° C. (decomp.), or a pharmaceutically acceptable acid addition salt thereof.

RELATED APPLICATIONS

The present application is a divisional application of application Ser.No. 07/826,232, filed Jan. 23, 1992, now U.S. Pat. No. 5,364,872, issuedNov. 15, 1994 which is a continuation of application Ser. No. 07/600,130filed Oct. 17, 1990, now abandoned, which is a continuation of Ser. No.07/478,724 filed Feb. 9, 1990, now abandoned, which is a continuation ofSer. No. 07/296,919, filed Jan. 11, 1989, now abandoned, which in turnis a continuation of Ser. No. 06/945,168 filed Dec. 22, 1986, nowabandoned, which in turn is a continuation of U.S. Ser. No. 06/723,043,filed Apr. 15, 1985, now abandoned.

FIELD OF THE INVENTION

This invention relates to diastereoisomer A of YM-09730, dextro-rotatoryoptical isomer thereof, and the pharmaceutically acceptable acidaddition salts thereof. The invention further relates to a process ofproducing these compounds and also to medicaments using these compoundsas an effective component.

BACKGROUND OF THE INVENTION

YM-09730 is a dihydropyridine-3,5-dicarboxylic acid ester derivativeshown by the following chemical structure, the chemical name of which is2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid3-(1-benzylpyrrolidin-3-yl)ester 5-methyl ester. ##STR2##

YM-09730 is the compound first synthesized by the researchers of thesame company as the applicant of this application and it is reportedthat the compound has a vasodilating activity and a hypotensiveactivity, and also prolonged effect of about these activities (U.S. Pat.No. 4,220,649; U. K. Patent No. 2,014,134, etc.,).

YM-09730 has two asymmteric carbon atoms and it is assumed from thestereochemical view point that there are isomers based on theseasymmeteric carbon atoms but there are no descriptions about theseisomers in the above-described patents and the existence of theseisomers was not confirmed.

SUMMARY OF THE INVENTION

The inventors have first succeeded in separating diastereoisomers A andB of YM-09730 and the optical isomers of them and have discovered thatisomer A (the diastereoisomer A and the dextro-rotatory optical isomerthereof are together referred to as isomer A unless specificallyindicated) has excellent specific pharmaceutical effects as compared toisomer B (the diastereoisomer B and the optical isomer thereof aretogether referred to as isomer B unless specifically indicated or to amixture (YM-09730) of both isomers A and B and the inventors havesucceeded in attaining the present invention based on the discovery. Inthis case, the melting point of the hydrochloride of the diastereoisomerA is 200° to 206° C. (decomp.) and the melting point of thehydrochloride of the dextrorotatory optical isomer thereof is 223° to230° C. (decomp.). Accordingly, the subject of the compounds of thisinvention is isomer A, specified by the melting point of thehydrochloride thereof, of YM-09730 and the pharmaceutically acceptableacid addition salts,

Thus, according to this invention, there is provided isomer A of2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid3-(1-benzylpyrrolidin-3-yl)ester 5-methyl ester, the melting point ofthe hydrochloride of diastereoisomer A being 200° to 206° C. (decomp.),and the pharmaceutically acceptable acid addition salts thereof.

DETAILED DESCRIPTION OF THE INVENTION

Now, the phamaceutically acceptable acid addition salts in thisinvention include malonates, oxalates, p-nitrobenzoates,2-ketoglutarates, maleates, 1-malates, hydrochlorides, sulfates,p-toluenesulfonates, phosphates and succinates.

As shown in the phrmacological experimental results describedhereinafter, the isomer A of this invention or the pharmaceuticallyacceptable acid. addition salts thereof show an area ratio of 15 to 38times that of diastereoisomer B and of 14 to 35 times that of anequivalent mixture of these diastereoisomers in the increasing rate ofan amount of a coronary blood stream by the direct administration intothe coronary arteries, which shows a high affinity of isomer A of thisinvention for the coronary arteries.

On the other hand, isomer B showed almost the same effect as that of theequivalent mixture of the isomers. This means that the pharmacologicalaction of YM-09730 is not a simple mean value of the physical mixtureratios of these isomers. Also, it is a new pharmacological discoveryfound by the inventors that isomer A and the pharmaceutically acceptableacid addition salts thereof have a high affinity for coronary arteriesand increase the possibility of the utilization of the compounds of thisinvention as medicaments.

Then, the production process for isomer A of this invention or thepharmaceutically acceptable acid addition salts thereof will beexplained below.

I. Production of diastereoisomer A

As described above, there are no descriptions about the isomers ofYM-09730 in aforesaid U.S. Pat. No. 4,220,649 and U. K Patent No.2,014,134 and the production of these isomers was not reported in anyreports published thereafter. A pair of diasetereoisomers differ inabsolute value of rotatory power as well as in the whole physical andchemical properties different from general optical antipodes and henceif these properties are not clarified, each isomer cannot be producedbased on the difference in these properties.

The inventors prepared YM-09730 by the Hantzch's synthesis process ofdihydropyridine (Ann. Chim., 215, 1(1882)) shown in Reference exampledescribed hereinafter and could know that YM-09730 was an equivalentmixture of diastereoisomer A and diastereoisomer B. Also, as the resultof various investigations on the process of obtaining diastereoisomer Afrom the mixture of the isomers based on this discovery, diastereoisomerA could be obtained by the following procedures.

(a) The mixture of the diastereoisomers was subjected to columnchromatography using silica gel as the carrier and a mixed solution ofethyl acetate and acetic acid as the eluent and diastereoisomer A wasobtained from the initial eluate and diastereoisomer B from the lattereluate. These two isomers are obtained from novel compounds firstdiscovered by the inventors.

As silica gel for use in the production process as a carrier for columnchromatography, any silica gel which is usually used for columnchromatography can be used without any restriction. Also, there is noparticular restriction about the mixing ratio of ethyl acetate andacetic acid in the mixed solvent for use as the eluent but usually themixed solvent containing a small amount of acetic acid is used. Themixing ratio is 30 to 50 v/v for ethyl acetate and about 1 to 10 v/v foracetic acid and if the content of acetic acid is lowered, the elutingtime of the desired compound is prolonged. The elution rate and theprocessing temperature can be properly selected.

(b) Also, in other process than the above process, the inventorssucceeded in producing the acid addition salt of diastereoisomer A byinducing the mixture of the distereoisomers to specific acid additionsalts and subjecting the mixture of the salts to a fractionalrecrystallization.

The acid addition salts for use in the production process are malonate,p-nitrobenzoate, maleate, etc.

These acid addition salts are crystalline salts and the solubility in anorganic solvent differs between diastereoisomer A and diasetereoisomerB. Thus, these properties can be utilized for producing diasetereoisomerA by a fractional recrystallization. A particularly suitable acidaddition salt is malonate. In the case of using the malonate, crystalshaving a very high content of diastereoisomer A can be obtained by onerecrystallization. As a solvent for use in the production process, thereare methanol, ethanol, acetone, acetonitrile, etc.

The acid addition salt of diastereoisomer A of YM-09730 obtained by theabove-described process can be acid-exchanged into a desired acidaddition salt by once inducing the salt into a free form and reactingwith other acid.

II. Production of dextro-rotatory optical isomer A

(a) The dextro-rotatory optical isomer of diastereoisomer A can beobtained by reacting the mixed free bases of diastereoisomers A and B orthe free base of the acid addition salt of diastereoisomer A of YM-09730obtained by the production process I described above with L-(-)-malicacid and then subjecting the product to optical resolution by anordinary manner as described hereinafter.

(b) Also, in a more preferred production process for the dextro-rotatoryoptical isomer of diastereoisomer A, dextro-rotatory optical isomer isseparated from YM-09730 (a mixture of the dextro-rotatory optical isomerof diastereoisomer A and the levo-rotatory optical isomer ofdiastereoisomer B) shown by formula (I) ##STR3## (wherein the wavelikebond means an α-bond or β-bond and the bond shown by the dense arrowmeans a β-bond), wherein the bond at 3-position of the pyrroldine ringis a specific β-bond.

The raw material compound (I) can be produced

(i) by reacting m-nitrobenzaldehyde shown by formula (II) ##STR4##(S)-3-acetoacetoxy-1-benzylpyrrolidine shown by formula (III) ##STR5##and 3-aminocrotonic acid methyl ester shown by formula (IV) ##STR6##(ii) by reacting m-nitrobenzaldehyde shown by formula (II) above,acetoacetic acid methyl ester shown by formula (V)

    CH.sub.3 COCH.sub.2 COOCH.sub.3                            (V)

and (S)-(-)-3-(3-aminocrotonoyloxy)-1-benzylpyrrolidine shown by formula(VI) ##STR7## (iii) reacting(S)-(-)-1-benzyl-3-[2-(m-nitrobenzylidene)-acetoacetoxy]pyrrolidineshown by formula (VII) ##STR8## obtained by previously reactingm-nitrobenzaldehyde shown by formula (II) above and(S)-3-acetoacetoxy-l-benzylpyrrolidine shown by formula (III) above and3-aminocrotonic acid methyl ester shown by formula (IV) above; or

(iv) reacting 2-(m-nitrobenzylidene)acetoacetic acid methyl ester shownby formula (VIII) ##STR9## obtained by previously reactingm-nitrobenzaldehyde shown by formula (II) above and acetoacetic acidmethyl ester shown by formula (V) above and(S)-(-)-3-(3-aminocrotonoyloxy)-l-benzylpyrrolidine shown by formula(VI) above.

These reaction proceed without use of solvent but it is advantageous toperform the reaction in a solvent which does not take part in thereaction, such as an alcohol, dioxane, dimethylformamide,dimethylsulfoxide, acetonitrile, water, etc. The reaction is performedby heating a mixture of an almost equimolar amount of each component.

In addition, the compound of formula (IV) or the compound of formula(VI) described above can be obtained by reacting the compound of formula(V) or the compound of formula (III) with ammonium acetate and aceticacid in benzene and azeotropically dehydrating the product. Also, thecompound of formula (IV) or compound of formula (VI) thus obtained issupplied to reaction (i) or (ii) described above after being isolated orwithout being isolated from the reaction mixture.

Also, the compound of formula (VII) or the compound of formula (VIII)which is the reaction product in the first step reaction process ofprocess (iii) or (iv) can be supplied to the last step process afterbeing isolated once or without being isolated.

The mixture of the dextro-rotatory optical isomer of diastereoisomer Aof YM-09730 and the levo-rotatory optical isomer of diastereoisomer Bthus obtained is subjected to column chromatography using silica gel asthe carrier and a mixture of ethyl acetate and acetic acid as the eluentto separate the dextro-rotatory optical isomer of diastereoisomer A ofYM-09730, or the mixture described above is reacted with L-(-)-malicacid to form the mixture of the L-(-)-malate of the dextro-rotatoryoptical isomer of diastereoisomer A and the L-(-)-malate of thelevo-rotatory optical isomer of diastereoisomer B and by fractionallyrecrystallizing the mixture, the L-(-)-malate of the dextro-rotatoryoptical isomer of diastereoisomer A can be obtained.

In the separation by column chromatography, the dextro-rotatory opticalisomer of diastereoisomer A is obtained from the first eluate and thelevo-rotatory optical isomer of diastereoisomer B can be obtained fromthe latter eluate.

As silica gel for use as the carrier, any silca gel which is generallyused for column chromatography can be used without particularrestriction. There is no particular restriction about the mixing ratioof ethyl acetate and acetic acid is for use as the eluent but the mixedsolvent containing a small amount of acetic acid preferred. It isadvantageous that the mixing ratio is 30 to 50 v/v for ethyl acetate andabout 1 to 10 v/v for acetic acid and when the content of acetic acid isfurther lowered, the eluting time for the desired compound is prolonged.

The eluting time and the processing temperature may be properlyselected.

On the other hand, the process of using L-(-)-malic acid can alsoutilized for the separation of the dextro-rotatory optical isomer ofdiastereoisomer A by recrystallization since the L-(-)-malate of thedextro rotatory optical isomer of diastereoisomer A of YM-09730 iscrystalline. As the solvent for use in the fractional recrystallization,there are methanol, ethanol, acetone, acetonitrile, etc.

The L-(-)-malate of the dextro-rotatory optical isomer ofdiastereoisomer A thus obtained can be used for medicament as it is, butcan be induced into the acetate or other suitable salt, if necessary, bytreating the L-(-)-malate with a base to form a free form and treatingthe product with a proper acid.

In addition, by hydrolyzing the levo-rotatory optical isomer ofdiastereoisomer B separated in the above process,(S)-(-)-1-benzyl-3-hydroxypyrrolidine can be recovered and hence thepyrrolidine can be reused as the raw material for producing the compoundof formula (I).

The pharmacological activities, acute toxicity and clinical doses ofisomer A are explained as follows.

(1) Coronary vasodilating effect in anesthetized dogs

In open-chest dogs anesthetized with 30 mg/kg i.v. of pentobarbitalsodium, arterial blood from the carotid artery was led to circumflexbranch of the left coronary artery by a extracorporeal loop. Aservocontrolled pum (model 1215D, Harvard Apparatus) was incorporated inthe circuit to maintain a constant perfusion pressure of 120 mmHg bymeans of a pump controller (SCS-22, Data Graph Co., Tokuya Tukada etal., Folia Pharmacol. Japon, 74, 59p, 1978). An electromagnetic flowprobe of extracorporeal type (MF-25, Nihon Koden) was also inserted inthe circuit to record coronary blood flow. A dose of 1 μg of thecompound was administered directly into the coronary artery and thencoronary blood flow was monitored until the blood flow returned to thepretreatment value. And then, the area under the percent increase incoronary blood flow after intracoronary injection of 1 μg of thecompound was calculated and used as an index of the overall increase incoronary blood flow. Results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Coronary vasodilating activity in anesthetized dogs                                       Overall increase                                                              in coronary blood                                                                           Duration of                                                     flow at 1 μg (%, min.)                                                                   the action                                          ______________________________________                                        Diastereoisomer A (dl                                                                       1816 ± 377   60                                              form) hydrochloride                                                           Diastereoisomer A (d                                                                        4559 ± 894   120                                             form) hydrochloride                                                           Diastereoisomer A (l                                                                        120 ± 21      5                                              form) hydrochloride                                                           Diastereoisomer B (dl                                                                       120 ± 28      5                                              form) hydrochloride                                                           Equivalent mixture of                                                                       129 ± 47     10                                              the hydrochlorides of                                                         diastereoisomers A (dl                                                        form) and B (dl form)                                                         ______________________________________                                    

When directly administered into the coronary artery, the overallincrease in coronary blood flow after 1 μg of the isomer A hydrochloridewas approximately 15 to 38 times higher than those after an identicaldose of the isomer B hydrochloride and the equivalent mixture of theseisomers, indicating that the isomer A hydrochloride possesses a highaffinity for the coronary artery. Furthermore, duration of the coronaryvasodilating activity after the isomer A hydrochloride was clearlylonger than those after the isomer B hydrochloride and the equivalentmixture of these isomer. Such a high affinity for the coronary arteryand long durability indicate that the isomer A hydrochloride is usefulfor the treatment of coronary artery disease such as angina pectoris.

(2) Hypotensive effect in anesthetized rats

Blood pressure was measured in urethane-anesthetized rats. The compoundwas intravenously administered in an increasing dose manner at aninterval of 20 minutes. The doses of the compounds required to lowermean blood pressure by 30 mmHg (ED 30 mmHg) was calculated from thedose-response curves and summarized in Table 2.

                  TABLE 2                                                         ______________________________________                                        Hypotensive activity in anesthetized rats                                                       ED30 mmHg                                                                     (mg/kg i.v.)                                                ______________________________________                                        Diastereoisomer A (dl form)                                                                       0.002                                                     hydrochloride                                                                 Diastereoisomer B (dl form)                                                                       0.14                                                      hydrochloride                                                                 ______________________________________                                    

As can be seen in Table 2, the hypotensive activity of thediastereoisomer A hydrochloride was about 70 times more potent than thatof the diastereoisomer B hydrochloride.

(3) Acute toxicity in mice

Six weeks old and male ICR mice weighing 27 to 29 g were used. Thecompound was suspended in 0.5% methylcellulose solution and administeredorally to mice. The LD₅₀ values of the compounds were calculated by themethod of Litchfield & Wilcoxon (Journal of Pharmacol. & Exp. Therap.,96, 99-113, 1949) and summarized in Table 3.

                  TABLE 3                                                         ______________________________________                                        Acute toxicity in mice                                                                         LD.sub.50 mg/kg p.o.                                         ______________________________________                                        Diastereoisomer A (dl form)                                                                      295 (242-360)                                              hydrochloride                                                                 Diastereoisomer A (d form)                                                                       190 (158-228)                                              hydrochloride                                                                 ______________________________________                                    

(4) Clinical doses

The clinical doses of the compounds of this invention depend on the bodyweight and the condition of disease of patients. The optimal doses areusually 0.1 to 2 mg for intravenous injection and 5-20 mg once or twicea day for oral administration.

Then, the compounds of this invention, the production process thereof,and the medicaments using these compounds are explained by the followingexamples and formulation examples. In addition, the production examplesof a mixture of diastereoisomer A and diastereoisomer B which is used asthe raw material is explained as Reference Example 1 and the productionexample of (S)-(-)-1-benzyl-3-hydroxypyrrolidine which is also used asthe raw material in the examples is explained as Reference Examples 2-4.

REFERENCE EXAMPLE 1

In 5 ml of isopropanol were dissolved 1.51 g (0.01 mole) of3-nitrobenzaldehyde, 2.61 g (0.01 mole) of1-benzyl-3-acetoacetoxypyrrolidine, and 1.15 g (0.01 mole) of3-aminocrotonic acid methyl ester and the solution thus obtained wasrefluxed for 8 hours. Then, the solvent was distilled off under reducedpressure, the residue thus formed was dissolved in chloroform, thesolution was washed, in succession, with diluted hydrochloric acid,water, a saturated aqueous sodium hydrogencarbonate solution, and driedover anhydrous magnesium sulfate. Then, the solvent was distilled offunder reduced pressure to provide 4.91 g of2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid3-(1-benzylpyrrolidin-3-yl)ester 5-methyl ester as a caramel form.

The formation ratio of the diastereoisomers thus obtained as the crudebase was analyzed by reversed-phase ion-pair high-performance liquidchromatography (HPLC) under the conditions shown below. The ratio ofdiastereoisomer A (retention time 28 min.): diastereoisomer B (retentiontime 29 min.) was 1:1.

Column: 4.6 mm×300 mm packed with Nucleosil® 5 C₁₈, column temperature:30° C., mobile phase: acetonitrile-0.05 mole potassiumdihydrogenphosphate (pH 3) (20:80 v/v) containing tetra-n-pentylammoniumbromide (3m mole) as counter ion, flow rate: 0.9 ml/min., monitor:ultraviolet detector (λ 254 nm).

In addition, by nuclear magnetic resonance analysis, diastereoisomer Aand diastereoisomer B were confirmed to have a different chemical shiftwith respect to methylene protons of N-benzyl group of YM-09730. Bymeasurement in heavy methanol d₄ (CD₃ OD) with Jeol NMR-spectrometerFX-90Q, isomer A and isomer B show the singlet signals corresponding totwo protons at 4.40 ppm and 4.30 ppm, respectively.

REFERENCE EXAMPLE 2

(1) In 66 ml of acetone were dissolved 17.7 g ofdl-1-benzyl-3-hydroxypyrrolidine and 15.2 g of D-(-)-mandelic acid withheating and the solution was allowed to stand overnight at 4° C. todeposit crystals. Then, 8.5 g of the crystals thus deposited werecollected and recrystallized from 26 ml of acetone to provide 5.1 g ofD-(-) -mandelate of (S)-(-)-1-benzyl-3-hydroxypyrrolidine. The specificrotation [α]_(D) ²⁰ was -45.5° C. (c=1, methanol). When therecrystallization was further repeated, the specific rotation was notchanged. The melting point was 101°-102° C.

By nuclear magnetic resonance analysis signal of N-CH₂ -Ph thus obtainedmandelate of (S)-(-)-form compound was observed at 4.03 ppm (singlet,2H). The signal of AB-type quartet (J=12.5 Hz) at 4.01 ppm of the(R)-(-) form was not observed.

(2) In 50 ml of chloroform was dissolved 22 g of D-(-)-mandelate of(S)-(-)-1-benzyl-3-hydroxypyrrolidine and the chloroform solution thusformed was washed with a solution of 14.4 g of sodium carbonate in 90 mlof water and dried over anhydrous magnesium sulfate. Then, afterdistilling off chloroform, the residue was distilled under reducedpressure to provide 11.5 g of S-(-)-1-benzyl-3-hydroxypyrrolidine. Theboiling point was 109° C./0.65 mmHg and [α]_(D) ²⁰ was -3.77° (c=5,methanol).

REFERENCE EXAMPLE 3

By reacting 75 g of (S)-(-)-malic acid and 75 ml of benzylamine for 3hours at 170° C., 52.7 g of (S)-(-)-1-benzyl-3-hydroxy-succinimide(melting point 99°-101° C., specific rotation [α]_(D) ²⁰ -51.1°, (c=1,methanol)) was obtained. In 340 ml of anhydrous tetrahydrofuran wassuspended 9.73 g of lithium aluminum hydride and a solution of 20.5 g ofthe above-described imide in 200 ml of anhydrous tetrahydrofuran wasadded dropwise to the suspension under ice-cooling. After refluxing themixture for 3 hours, 100 g of sodium sulfate decahydrate was added tothe mixture under ice-cooling and the resultant mixture was stirredovernight under ice-cooling. Insoluble solids were filtered off, thesolvent was evaporated under reduced pressure from the filtrate, and theresidue was distilled under reduced pressure to provide 13.8 g of(S)-(-)-1-benzyl-3-hydroxypyrrolidine having a boiling point of 109° to115° C./0.8 mmHg and specific rotation [α]_(D) ²⁰ of -3.0° (c=5,methanol). It was confirmed that the (S)-(-)-form obtained abovecontained 10% R-(+)-1-benzyl-3-hydroxypyrrolidine by proton nuclearmagnetic resonance analysis of the 3-position using a shift reagentEu-TFMC(III). The product was induced to the D-(-)-mandelate asReference Example 2. The mandelate [α]_(D) ²⁰ -45.2° (c=1, MeOH)obtained by recrystallizing from 3 times by volume of ethanol and then 6times by volume of ethanol-toluene (1:5 v/v), was treated withchloroform and a solution of aqueous sodium carbonate as ReverenceExample 2 to give 8.6 g of (S)-(-)-1-benzyl-3-hydroxypyrrolidine;boiling point 115° to 120° C./1.2 to 1.5 mmHg, [α]_(D) ²⁰ -3.77° (c=5,methanol).

REFERENCE EXAMPLE 4

To 50 ml of 9-borabicyclo[3,3,1]nonane (0.5M tetrahydrofuran solution)was added 3.4 g of 2-(-)-pinene and the mixture was stirred for 5 hoursat 60° C. After cooling the mixture to room temperature, 1.75 g of1-benzyl-3-pyrrolidinone was added thereto. After stirring the mixturefor 4 days at room temperature, 1.3 ml of acetaldehyde was added theretoat 0° C. Then, the solvent was distilled off under reduced pressure fromthe reaction mixture and 20 ml of ether was added to the rescue. Aftercooling the mixture to 0° C., 1.5 ml of 2-aminoethanol was added theretoand the resultant mixture was stirred. Precipitates thus formed werefiltered off . The ether solution recovered as the filtrate wasextracted with 1N hydrochloric acid and the hydrochloric acid layer thusformed was made basic with sodium carbonate and extracted withdichloromethane. The extract thus formed was dried by anhydrousmagnesium sulfate and concentrated to provide 1.1 g of a crude product.Then, by subjecting the crude product to distillation under reducedpressure, 0.6 g of a purified product was obtained. The boiling pointthereof 106° C./0.9 mmHg. (S)-(-)-1-benzyl-3-hydroxypyrrolidine thusobtained was 30% e. e. analyzed by the nuclear magnetic resonancespectra with respect to the proton at the 3-position using a shiftreagent Eu-TFMC(III).

PRODUCTION EXAMPLE OF DIASTEREOISOMER A Example 1

In 25 ml of chloroform was dissolved 4.91 g of the crude free base ofYM-09730 obtained in Reference Example 1 and after adding thereto 15 mlof 10% hydrochloric acid followed by stirring well, the organic phasethus formed was separated. The organic layer was treated again by thesame manner as above with 10 ml of 10% hydrochloric acid, and afterdrying the treated product with anhydrous magnesium sulfate, the solventwas distilled off under reduced pressure. The residue thus obtained wasdissolved in 10.4 ml of acetone and the solution was allowed to stand toprovide 3.5 g of the hydrochloride of YM-09730 as crystals. The productwas disssolved in 1.8 ml of methanol and recrystallized with theaddition of 8 ml of acetone. By repeating once, 2.38 g of thehydrochloride of YM-09730 was obtained. The ratio of diastereoisomerA:diastereoisomer B in the hydrochloride was 65.6:34.4 by HPLC. In 25 mlof chloroform was dissolved 2.15 g of the aforesaid salt, the solutionthus obtained was washed twice each time with 15 ml of a saturatedaqueous sodium hydrogencarbonate solution, and the organic layer thusformed was collected and dried over anhydrous magnesium sulfate. Then,the solvent was distilled off under reduced pressure and 2 g of a freebase thus obtained was subjected to silica gel column chromatography(column: LiChroprep® Si60, C size, Eluent ethyl acetate-acetic acid=30:5v/v) to provide the oily acetate of diastereoisomer A.

The product was dissolved in 10 ml of chloroform, the solution thusformed was washed, in succession, with 10 ml of a saturated aqueoussodium hydrogencarbonate solution, 10 ml of water, and 10 ml of 10%hydrochloric acid, and dried with anhydrous magnesium sulfate. Then, thesolvent was distilled off under reduced pressure and the residue thusobtained was treated with 0.8 ml of acetone to provide 0.4 g of thehydrochloride of diastereoisomer A.

Example 2

In 15 ml of acetonitrile were dissolved 4.91 g of the crude free base ofYM-09730 obtained in Reference Example 1 and 1.04 g of malonic acid andthe solution thus obtained was allowed to stand overnight at 0° to 5° C.Crystals thus formed were collected by filtration, washed with a smallamount of cold acetonitrile to provide 2.03 g of the malonate ofYM-09730. (Ratio of diastereoisomer A:B was 89.1:10.9). The product wasrecrystallized twice each time from 25 times by volume of methanol toprovide 1.0 g g of the malonate of 100% diastereoisomer A of YM-09730.

Example 3

In 15 ml of methanol were dissolved 4.91 g of the crude free base ofYM-09730 obtained in Reference Example 1 and 1.04 g of malonic acid byheating and the solution thus formed was allowed to stand overnight at0° to 5° C. Crystals thus deposited were collected by filtration, washedwith methanol, and dried under reduced pressure to provide 1.88 g of themalonate of YM-09730. The ratio of diastereoisomers A:B of the crystalsthus obtained was 90.7:9.3. The crystals were recrystallized twice frommethanol to provide the malonate of diasetereoiosmer A containing noisomer B.

Example 4

By following the same procedure as in Example 3 using acetonitrile inplace of methanol, 2.03 g of a malonate was obtained. The ratio ofdiastereoisomers A:B was 89.1:10.9. The product was recrystallized from25 times by volume of methanol to provide 1.57 g of the malonate ofdiastereoisomers A and B at a ratio of 99.5:0.5. Furthermore, byrecrystallizing the product from 25 times by volume of methanol, 1.27 gof the maolonate of YM-09730 wherein the presence of diastereoisomer Bwas not detected by high performance liquid chromatography was obtained.In 5 ml of chloroform was suspended 1.27 g of the malonate, thechloroform suspension was treated, in succession, twice each time with asaturated aqueous sodium carbonate solution, twice each time with 2.5 mlof water, and then twice each time with 2.5 ml of 10% hydrochloric acid,the chloroform solution thus washed was dried over anhydrous magnesiumsulfate, filtered, and evaporated to dryness under reduced pressure. Theresidue thus formed was dissolved in 2 ml of acetone and the solutionwas allowed to stand to deposit 1.09 g of the hydrochloride ofdiastereoisomer A of YM-09730.

Example 5

In 5 ml of chloroform was suspended 595 mg of the malonate ofdistereoisomer A of YM-09730 and the suspension thus obtained wastreated twice each time with 2.5 ml of a saturated aqueous sodiumhdyrogen carbonate solution and then twice each time with 5 ml of water.The chloroform solution thus obtained was dried over anhydrous magnesiumsulfate and evaporated to dryness under reduced pressure to provide 491mg of the free base of diastereoisomer A as a caramel. The product and126 mg of oxalic acid dihydrate were dissolved in 3 ml of acetone, thesolution thus formed was allowed to stand at 4° C., and the crystalsthus deposited were collected by filtration to provide 400 mg of theoxalate of diastereoisomer A of YM-09730.

Example 6

By following the same procedure using 146 mg of 2-ketoglutaric acid inplace of oxalic acid in Example 5, 250 mg of the 2-ketoglutarate ofdiastereoisomer A of YM-09730 was obtained.

Example 7

By following the same procedure using 167 mg of p-nitrobenzoic acid inplace of oxalic acid in Example 5, 530 mg of p-nitrobenzoate ofdiastereoisomer A of YM-09730 was obtained.

Example 8

By following the same procedure as in Example 5 using 116 mg of maleicacid in place of oxalic acid, 300 mg of the maleate of diastereoisomer Aof YM-09730 was obtained.

Example 9

In 2 ml of acetone was dissolved 491 mg of diastereoisomer A of 09730and after adding thereto 1 ml of a methanol solution of 1 mole ofphosphoric acid, the solution was allowed to stand at 4° C. Crystalsthus deposited were collected by filtration to provide 480 mg of thephosphate of diastereoisomer A of YM-09730.

The properties of the desired compounds obtained in Examples 1 to 9 areshown in the following table.

    __________________________________________________________________________                                   Properties Elemental analysis                  Desired Compound                                                                        Composition Formula                                                                          M. P. C (%)                                                                             H (%)                                                                             N (%)                                                                             Cl (%)                             __________________________________________________________________________    Phosphate C.sub.27 H.sub.29 N.sub.3 O.sub.6 . H.sub.3 PO.sub.4 . 1/2H.sub.              2 O            216-218                                                                             54.39                                                                             5.56                                                                              6.96                                                                  54.18                                                                             5.56                                                                              7.02                                   Hydrochloride                                                                           C.sub.27 H.sub.29 N.sub.3 O.sub.6 . HCl                                                      203-205                                                                             61.59                                                                             5.71                                                                              8.08                                                                              6.90                                                        (decomp.)                                                                           61.42                                                                             5.73                                                                              7.96                                                                              6.71                               p-Nitrobenzoate                                                                         C.sub.27 H.sub.29 N.sub.3 O.sub.6 . C.sub.7 H.sub.5 NO.sub.4 .                1/4H.sub.2 O   150-151                                                                             61.58                                                                             5.20                                                                              8.32                                                                  61.58                                                                             5.24                                                                              8.32                                   Maleate   C.sub.27 H.sub.29 N.sub.3 O.sub.6 . C.sub.4 H.sub.4 O.sub.4                                  168-169                                                                             61.40                                                                             5.49                                                                              6.85                                                                  61.28                                                                             5.47                                                                              6.92                                   2-Ketoglutarate                                                                         C.sub.27 H.sub.29 N.sub.3 O.sub.6 . C.sub.5 H.sub.4 O.sub.3                                  160-161                                                                             60.36                                                                             5.48                                                                              6.52                                                                  60.28                                                                             5.53                                                                              6.59                                   Oxalate   C.sub.27 H.sub.29 N.sub.3 O.sub.6 . C.sub.2 H.sub.2 O.sub.                                   179-180                                                                             60.14                                                                             5.62                                                                              6.93                                                                  60.36                                                                             5.70                                                                              6.88                                   Malonate  C.sub.27 H.sub.29 N.sub.3 O.sub.6 . C.sub.3 H.sub.4 O.sub.4                                  181.5-182.5                                                                         59.89                                                                             5.37                                                                              7.23                                                            (decomp.)                                                                           60.50                                                                             5.58                                                                              7.06                                                                  (Upper: Found values)                                                         (Lower: Calculated values)                     __________________________________________________________________________

Hydrochloride: NMR (in CD₃ OD, TMS internal standard, δppm)

1.80-2.70 (2H, broad m, C₄ '--H₂)

2.32,2.34 (6H, s, C₂,6 --CH₃)

3.0-4.0 (4H, m, C_(2'),5' --H₂)

3.63 (3H, s --COOCH₃)

4,40 (2H, s, --CH₂ -- φ)

5.08 (1H, s, C₄ --H)

5.30 (1H, m, C_(3') --H)

7.30-8.20 (9H, m, H of benzene ring) ##STR10##

Example 10

In 5 ml of chloroform was suspended 1.5 g of the malonate ofdiastereoisomer A obtained in Example 3 or 4 and the suspension wastreated, in succession, twice each time with 3 ml of a saturated aqueoussodium hydrogen carbonate solution and then twice each time with 3 ml ofwater. The chloroform solution was dried over anhydrous magnesiumsulfate and then the solvent was distilled off under reduced pressure.To the residue thus obtained was added 6 ml of ethanol and the mixturewas allowed to stand overnight at 5° C. to obtain 0.86 g of crystals ofthe free base of diastereoisomer A of YM-09730.

Melting point 145° to 148° C.

    ______________________________________                                        Elemental analysis for (C.sub.27 H.sub.29 N.sub.3 O.sub.6)                             C (%)      H (%)   N (%)                                             ______________________________________                                        Calculated:                                                                              65.98        5.95    8.55                                          Found:     66.04        6.00    8.53                                          ______________________________________                                    

NMR (in CDCl₃, TMS internal standard, δppm)

1.40 to 2.96 (6H, m, C_(2'4'),5' --H₂)

2.34, 2.36 (6H, s, C₂,6 --CH₃)

3.65 (5H, s, --COOCH₃ and --CH₂ φ)

5.10 (1H, s, C₄ --H)

5.12 (1H, m, C_(3') --H)

5.78 (1H, broad s, NH)

7.18 to 8.25 (9H, m, H of benzene ring) ##STR11##

PRODUCTION OF DEXTRO-ROTATORY A ISOMER Example 11

(1) In 15 ml of acetonitrile were dissolved 4.91 g of the crude freebase obtained in Reference Example 1 above and 1.04 g of malonic acidand the solution was allowed to stand overnight at 0° to 5° C. Crystalsthus deposited were collected by filtration (2.03 g) and recrystallizedtwice each time from 25 times by volume of methanol to provide 1.27 g ofthe malonate of diastereoisomer A of YM-09730 containing nodiastereoisomer B. The melting point thereof was 181.5° C. to 182.5° C.(decomp.). In 5 ml of chloroform was suspended 1.27 g of the malonatethus obtained and the suspension was washed, in succession, twice eachtime with 2.5 ml of a saturated aqueous sodium hydrogencarbonatesolution, once with 2.5 ml of water, and then twice each time with 2.5ml of an aqueous 10% hydrochloric acid solution. The chloroform solutionwas dried over anhydrous magnesium sulfate and evaporated to drynessunder reduced pressure. The residue thus formed was dissolved in 2 ml ofacetone, the solution was allowed to stand, and 1.09 g of thehydrochloride of diastereoisomer A of YM-09730 thus deposited wascollected.

(2) By treating 4.67 g of the hydrochloride of diastereoisomer A ofYM-09730 obtained in the above step with saturated aqueous sodiumhydrogencarbonate, 4.35 g of the free base thereof was obtained and then4.35 g of the free base thus obtained and 1.18 g of L-(-)-malic acidwere dissolved in 28 ml of ethanol with heating and the solution wasallowed to stand overnight at 0° to 5° C. Crystals thus deposited werecollected by filtration and dried to provide 2.43 g of the L-(-)-malateof the dextro-rotatory optical isomer of diastereoisomer A of YM-09730.The crystals were recrystallized from 85 ml of ethanol to provide 2.21 gof the L-(-)-malate. The specific rotation [α]_(D) ²⁰ was +82.2° (c=0.5,methanol). When the product was recrystallized from ethanol, the changeof the specific rotation was not observed.

Melting point 190° to 191° C (decomp.).

    ______________________________________                                        Elemental analysis (for C.sub.27 H.sub.29 N.sub.3 O.sub.6 . C.sub.4           H.sub.6 O.sub.5)                                                                       C (%)      H (%)   N (%)                                             ______________________________________                                        Found:     59.72        5.80    6.73                                          Calculated:                                                                              59.51        5.64    6.72                                          ______________________________________                                    

Example 12

In 25 ml of acetone were dissolved 4.91 g of the crude free base ofYM-09730 obtained in Reference Example 1 and 1.34 g of L-(-)-malic acidand the solution thus formed was stirred for 48 hours at 0° to 5° C.Crystals thus deposited were collected by filtration and washed with asmall amount of cold acetone to provide 0.57 g of the L-(-)-malate ofthe dextro-rotatory optical isomer of diastereoisomer A of YM-09730. Thespecific rotation [α]_(D) ²⁰ was +78.3° (c=0.5, metahnol). Also, thespecific rotation [α]_(D) ²⁰ of 0.44 g of the crystals obtained byrecrystallizing the product from 50 times by volume of ethanol was+82.2° (c=0.5, methanol).

Melting point 190° to 191° C. (decomp.).

Example 13

In 8 ml of chloroform was suspended 2.21 g of the L-(-)-malate of thedextro-rotatory optical isomer of diastereoisomer A of YM-09730 obtainedin Example 11 and the suspension was treated, in succession, twice eachtime with 4.3 ml of a saturated aqueous sodium hydrogen carbonatesolution, 4.3 ml of water, and then twice each time with 4.3 ml of 10%hydrochloric acid. The chlorofrom solution was dried over anhydrousmagnesium sulfate and after filtration, the solution was evaporated todryness under reduced pressure. The residue thus formed was dissolved in3.5 ml of acetone and the solution thus formed was allowed to stand toprovide 1.64 g of the hydrochloride of the dextro-rotatory opticalisomer of diastereoisomer A of YM-09730 as deposits.

The specific rotation [α]_(D) ²⁰ was +116.5° (c=0.5, methanol).

Melting point: 223° to 225° C. (decomp.)

    ______________________________________                                        Elemental analysis (for C.sub.27 H.sub.29 N.sub.3 O.sub.6 . HCl)                       C (%) H (%)      N (%)   Cl (%)                                      ______________________________________                                        Found:     61.35   5.55       8.01  6.96                                      Calculated:                                                                              61.42   5.73       7.96  6.71                                      ______________________________________                                    

NMR (in CD₃ OD, TMS internal standard, δppm)

1.80-2.70 (2H, broad m, C_(4') --H₂)

2.32, 2.34 (6H, s, C₂,6 --CH₃)

3.0-4.0 (4H, m, C_(2'),5' --H₂)

3.64 (3H, s, --COOCH₃)

4.42 (2H, s, --CH₂ φ)

5.08 (1H, s C₄ --H)

5.30 (1H, m, C_(3') --H)

7.30-8.20 (9H, m, H of benzene ring)

Example 14

In 5 ml of isopropanol were dissolved(S)-3-acetoacetoxy-1-benzylpyrroldine obtained by reacting 1.77 g of(S)-(-)-1-benzyl-3-hydroxypyrrolidine ([α]_(D) ²⁰ -3.77°, c=5, methanol)and 0.84 g of diketene for 3 hours at 70° to 80° C., 1.51 g ofm-nitrobenzaldehyde and 1.15 g of 3-aminocrotonic acid methyl ester, andthe solution was refluxed for 8 hours. The solvent was then distilledoff under reduced pressure from the reaction mixture. The residue thusformed was dissolved in chloroform, the solution thus formed was washed,in succession, with a diluted hydrochloric acid, water, and then asaturated aqueous sodium hydrogen carbonate solution, and dried overanhydrous magnesium sulfate. Then, the solvent was distilled off underreduced pressure to provide 4.91 g of a mixture of the dextro-rotatoryoptical isomer of diastereoisomer A and the levo-rotatory optical isomerof diastereoisomer B of YM-09730 as a caramel. The crude free base thusobtained was applied to silica gel column chromatography (column:Wakogel C-200, 2,000 g, eluent: ethyl acetate-acetic acid=6:1 v/v),whereby the oily acetate of the dextro-rotatory optical isomer ofdiastereoisomer A of YM-09730 showing a retention time of 28 min. byhigh-performance liquid chromatography.

The product was treated in chloroform with saturated aqueous sodiumhydrogen carbonate and then diluted hydrochloric acid to provide 1.68 gof the hydrochloride of the dextro-rotatory optical isomer ofdiastereoisomer A of YM-09730. The specific rotation [α]_(D) ²⁰ was+116.5° (c=0.5, methanol)

Example 15

In 25 ml of chloroform was dissolved 4.9 g of the crude free baseobtained as in Example 14 and after adding thereto 15 ml of 10%hydrochloric acid, the resultant mixture was stirred well. Then, theorganic layer thus formed was separated, washed again with 10 ml of 10%hydrochloric acid, and dried over anhydrous magnesium sulfate.Thereafter, the solvent was distilled off under reduced pressure, theresidue thus formed was dissolved in 10 ml of acetone, and the solutionwas allowed to stand for 2 days at 4° C. to provide 2.7 g of thehydrochloride by collecting the crystals thus deposited. For removingthe levo-rotatory optical isomer of diastereoisomer B therefrom, theproduct thus obtained was treated with saturated aqueous sodium hydrogencarbonate in chloroform to form a free base and after adding 0.68 g ofL-(-)-malic acid in 15 ml of ethanol, the resultant mixture was allowedto stand for 2 days at 4° C. Crystals thus deposited were collected byfiltration and recrystallized from ethanol to provide 1.33 g of theL-(-)-malate of the dextro-rotatory optical isomer of diastereoisomer Aof YM-09730 showing a retension time of 28 min. by high-performanceliquid chromatography. The specific rotation [α]_(D) ²⁰ was +82.1°(c=0.5, metnaol).

Example 16

In 5 ml of chloroform was suspended 1.25 g of the L-(-)malate of thedextro-ratotory optical isomer of diastereoisomer A of YM-09730 and thesuspension thus obtained was treated, in succession, twice each timewith 3 ml of saturated aqueous sodium hydrogen carbonate and then twiceeach time with 3 ml of water. The chloroform solution was dried overanhydrous magnesium sulfate and then the solvent was distilled off underreduced pressure. To the residue thus formed was added 6 ml of ethanoland the mixture was allowed to stand overnight at 5° C. to provide 0.84g of the crystals of the free base of the dextro-rotatory optical isomerof diastereoisomer A of YM-09730.

Melting point 138° to 140° C.

    ______________________________________                                        Elemental analysis (for C.sub.27 H.sub.29 N.sub.3 O.sub.6)                             C (%)      H (%)   N (%)                                             ______________________________________                                        Calculated:                                                                              65.98        5.95    8.55                                          Found:     66.04        5.96    8.51                                          ______________________________________                                    

NMR (in CDCl₃, TMS internal standard, δppm)

1.40 to 3.0 (6H, m, C_(2'),4'5' --H₂)

2.34, 2.36 (6H. s, C₂,6 --CH₃)

3.65 (5H, s, --COOCH₃ and --CH₂ φ)

5.10 (1H, s, C₄ --H)

5.12 (1H, m, C_(3') --H)

5.78 (1H, broad s, --NH)

7.16 to 8.24 (9H, m, H of benzene ring).

Formulation Example 1 (Tablet)

    ______________________________________                                                      One tablet                                                                             5,000 tabs.                                            ______________________________________                                        Isomer A hydrochloride                                                                        10.0 mg    50 g                                               Lactose         101.0 mg   502 g                                              Corn starch     25.3 mg    126.5 g                                            Hydroxypropyl cellulose                                                                       3.0 mg     15 g                                               Magnesium stearate                                                                            0.7 mg     3.5 g                                                              140 mg     700 g                                              ______________________________________                                    

To a uniform mixture of 50 g of the hydrochloride of isomer A, 502 g oflactose, and 126.5 g of corn starch was added 150 g of an aqueous 10%hydroxypropyl cellulose and the mixture was kneaded and granulated.After drying, 3.5 g of magnesium stearate was added to the granules andthey were uniformly mixed and then formed into tablets each of 140 mg.

Formulation Example 2 (Capsule)

    ______________________________________                                                      One capsule                                                                             1,000 cap.                                            ______________________________________                                        Isomer A hydrochloride                                                                        10.0 mg     10 g                                              Crystal lactose 189.0 mg    189 g                                             Magnesium stearate                                                                            1.0 mg      1 g                                                               200 mg      200 g                                             ______________________________________                                    

The above components were uniformly mixed and 200 mg thereof was filledin each capsule to provide capsule medicaments.

Formulation Example 3 (Injection)

    ______________________________________                                        Isomer A hydrochloride                                                                            1 mg                                                      D-sorbitol         100 mg                                                     ______________________________________                                    

The above components were dissolved in distilled water for injection andafter adjusting the pH thereof to 4 by the addition of hydrochloricacid, distilled water for injection was added to make the total volumeto 2 ml.

What is claimed is:
 1. A dextro-rotatory optical isomer ofdiastereoisomer A of(±)-2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylicacid 3-(1-benzylpyrrolidin-3-yl)ester 5-methyl ester having the formula##STR12## and wherein the melting point of the hydrochloride of saiddextrorotatory optical isomer is 223° to 230° C. (decomp.), or apharmaceutically acceptable acid addition salt thereof.
 2. An acidaddition salt as claimed in claim 1, wherein the acid addition salt isL-(-)malate.
 3. An acid addition salt as claimed in claim 1, wherein theacid addition salt is hydrochloride.